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Craig A Townsend - One of the best experts on this subject based on the ideXlab platform.

  • structure of a bound peptide phosphonate reveals the mechanism of Nocardicin bifunctional thioesterase epimerase hydrolase half reactions
    Nature Communications, 2019
    Co-Authors: Ketan D Patel, Nicole M Gaudelli, Craig A Townsend, Felipe B Dandrea, Andrew R Buller, Andrew M Gulick
    Abstract:

    Nonribosomal peptide synthetases (NRPSs) underlie the biosynthesis of many natural products that have important medicinal utility. Protection of the NRPS peptide products from proteolysis is critical to these pathways and is often achieved by structural modification, principally the introduction of D-amino acid residues into the elongating peptide. These amino acids are generally formed in situ from their L-stereoisomers by epimerization domains or dual-function condensation/epimerization domains. In singular contrast, the thioesterase domain of Nocardicin biosynthesis mediates both the effectively complete L- to D-epimerization of its C-terminal amino acid residue (≥100:1) and hydrolytic product release. We report herein high-resolution crystal structures of the Nocardicin thioesterase domain in ligand-free form and reacted with a structurally precise fluorophosphonate substrate mimic that identify the complete peptide binding pocket to accommodate both stereoisomers. These structures combined with additional functional studies provide detailed mechanistic insight into this unique dual-function NRPS domain.

  • β lactam formation by a non ribosomal peptide synthetase during antibiotic biosynthesis
    Nature, 2015
    Co-Authors: Nicole M Gaudelli, Darcie H Long, Craig A Townsend
    Abstract:

    Non-ribosomal peptide synthetases are giant enzymes composed of modules that house repeated sets of functional domains, which select, activate and couple amino acids drawn from a pool of nearly 500 potential building blocks. The structurally and stereochemically diverse peptides generated in this manner underlie the biosynthesis of a large sector of natural products. Many of their derived metabolites are bioactive such as the antibiotics vancomycin, bacitracin, daptomycin and the β-lactam-containing penicillins, cephalosporins and Nocardicins. Penicillins and cephalosporins are synthesized from a classically derived non-ribosomal peptide synthetase tripeptide (from δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase). Here we report an unprecedented non-ribosomal peptide synthetase activity that both assembles a serine-containing peptide and mediates its cyclization to the critical β-lactam ring of the Nocardicin family of antibiotics. A histidine-rich condensation domain, which typically performs peptide bond formation during product assembly, also synthesizes the embedded four-membered ring. We propose a mechanism, and describe supporting experiments, that is distinct from the pathways that have evolved to the three other β-lactam antibiotic families: penicillin/cephalosporins, clavams and carbapenems. These findings raise the possibility that β-lactam rings can be regio- and stereospecifically integrated into engineered peptides for application as, for example, targeted protease inactivators.

  • epimerization and substrate gating by a te domain in β lactam antibiotic biosynthesis
    Nature Chemical Biology, 2014
    Co-Authors: Nicole M Gaudelli, Craig A Townsend
    Abstract:

    Synthetic analogs of Nocardicin G—a key precursor of β-lactam antibiotics—are used to show that construction of this enigmatic modified tripeptide relies on an unusual thioesterase domain that epimerizes one residue of an intermediate pentapeptide, but only when the lactam ring is already formed.

  • non ribosomal propeptide precursor in Nocardicin a biosynthesis predicted from adenylation domain specificity dependent on the mbth family protein noci
    Journal of the American Chemical Society, 2013
    Co-Authors: Jeanne M Davidsen, David M Bartley, Craig A Townsend
    Abstract:

    Nocardicin A is a monocyclic β-lactam isolated from the actinomycete Nocardia uniformis that shows moderate antibiotic activity against a broad spectrum of Gram-negative bacteria. The monobactams are of renewed interest due to emerging Gram-negative strains resistant to clinically available penicillins and cephalosporins. Like isopenicillin N, Nocardicin A has a tripeptide core of non-ribosomal origin. Paradoxically, the Nocardicin A gene cluster encodes two non-ribosomal peptide synthetases (NRPSs), NocA and NocB, predicted to encode five modules pointing to a pentapeptide precursor in Nocardicin A biosynthesis, unless module skipping or other nonlinear reactions are occurring. Previous radiochemical incorporation experiments and bioinformatic analyses predict the incorporation of p-hydroxy-l-phenylglycine (l-pHPG) into positions 1, 3, and 5 and l-serine into position 4. No prediction could be made for position 2. Multidomain constructs of each module were heterologous expressed in Escherichia coli for d...

  • Non-ribosomal Propeptide Precursor in Nocardicin A Biosynthesis Predicted from Adenylation Domain Specificity Dependent on the MbtH Family Protein NocI
    2013
    Co-Authors: Jeanne M Davidsen, David M Bartley, Craig A Townsend
    Abstract:

    Nocardicin A is a monocyclic β-lactam isolated from the actinomycete Nocardia uniformis that shows moderate antibiotic activity against a broad spectrum of Gram-negative bacteria. The monobactams are of renewed interest due to emerging Gram-negative strains resistant to clinically available penicillins and cephalosporins. Like isopenicillin N, Nocardicin A has a tripeptide core of non-ribosomal origin. Paradoxically, the Nocardicin A gene cluster encodes two non-ribosomal peptide synthetases (NRPSs), NocA and NocB, predicted to encode five modules pointing to a pentapeptide precursor in Nocardicin A biosynthesis, unless module skipping or other nonlinear reactions are occurring. Previous radiochemical incorporation experiments and bioinformatic analyses predict the incorporation of p-hydroxy-l-phenylglycine (l-pHPG) into positions 1, 3, and 5 and l-serine into position 4. No prediction could be made for position 2. Multidomain constructs of each module were heterologous expressed in Escherichia coli for determination of the adenylation domain (A-domain) substrate specificity using the ATP/PPi exchange assay. Three of the five A-domains, from modules 1, 2, and 4, required the addition of stoichiometric amounts of MbtH family protein NocI to detect exchange activity. On the basis of these analyses, the predicted product of the NocA and NocB NRPSs is l-pHPG–l-Arg–d-pHPG–l-Ser–l-pHPG, a pentapeptide. Despite being flanked by non-proteinogenic amino acids, proteolysis of this pentapeptide by trypsin yields two fragments from cleavage at the C terminus of the l-Arg residue. Thus, a proteolytic step is likely involved in the biosynthesis of Nocardicin A, a rare but precedented editing event in the formation of non-ribosomal natural products that is supported by the identification of trypsin-encoding genes in N. uniformis

Nicole M Gaudelli - One of the best experts on this subject based on the ideXlab platform.

  • structure of a bound peptide phosphonate reveals the mechanism of Nocardicin bifunctional thioesterase epimerase hydrolase half reactions
    Nature Communications, 2019
    Co-Authors: Ketan D Patel, Nicole M Gaudelli, Craig A Townsend, Felipe B Dandrea, Andrew R Buller, Andrew M Gulick
    Abstract:

    Nonribosomal peptide synthetases (NRPSs) underlie the biosynthesis of many natural products that have important medicinal utility. Protection of the NRPS peptide products from proteolysis is critical to these pathways and is often achieved by structural modification, principally the introduction of D-amino acid residues into the elongating peptide. These amino acids are generally formed in situ from their L-stereoisomers by epimerization domains or dual-function condensation/epimerization domains. In singular contrast, the thioesterase domain of Nocardicin biosynthesis mediates both the effectively complete L- to D-epimerization of its C-terminal amino acid residue (≥100:1) and hydrolytic product release. We report herein high-resolution crystal structures of the Nocardicin thioesterase domain in ligand-free form and reacted with a structurally precise fluorophosphonate substrate mimic that identify the complete peptide binding pocket to accommodate both stereoisomers. These structures combined with additional functional studies provide detailed mechanistic insight into this unique dual-function NRPS domain.

  • β lactam formation by a non ribosomal peptide synthetase during antibiotic biosynthesis
    Nature, 2015
    Co-Authors: Nicole M Gaudelli, Darcie H Long, Craig A Townsend
    Abstract:

    Non-ribosomal peptide synthetases are giant enzymes composed of modules that house repeated sets of functional domains, which select, activate and couple amino acids drawn from a pool of nearly 500 potential building blocks. The structurally and stereochemically diverse peptides generated in this manner underlie the biosynthesis of a large sector of natural products. Many of their derived metabolites are bioactive such as the antibiotics vancomycin, bacitracin, daptomycin and the β-lactam-containing penicillins, cephalosporins and Nocardicins. Penicillins and cephalosporins are synthesized from a classically derived non-ribosomal peptide synthetase tripeptide (from δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase). Here we report an unprecedented non-ribosomal peptide synthetase activity that both assembles a serine-containing peptide and mediates its cyclization to the critical β-lactam ring of the Nocardicin family of antibiotics. A histidine-rich condensation domain, which typically performs peptide bond formation during product assembly, also synthesizes the embedded four-membered ring. We propose a mechanism, and describe supporting experiments, that is distinct from the pathways that have evolved to the three other β-lactam antibiotic families: penicillin/cephalosporins, clavams and carbapenems. These findings raise the possibility that β-lactam rings can be regio- and stereospecifically integrated into engineered peptides for application as, for example, targeted protease inactivators.

  • epimerization and substrate gating by a te domain in β lactam antibiotic biosynthesis
    Nature Chemical Biology, 2014
    Co-Authors: Nicole M Gaudelli, Craig A Townsend
    Abstract:

    Synthetic analogs of Nocardicin G—a key precursor of β-lactam antibiotics—are used to show that construction of this enigmatic modified tripeptide relies on an unusual thioesterase domain that epimerizes one residue of an intermediate pentapeptide, but only when the lactam ring is already formed.

Jeanne M Davidsen - One of the best experts on this subject based on the ideXlab platform.

  • non ribosomal propeptide precursor in Nocardicin a biosynthesis predicted from adenylation domain specificity dependent on the mbth family protein noci
    Journal of the American Chemical Society, 2013
    Co-Authors: Jeanne M Davidsen, David M Bartley, Craig A Townsend
    Abstract:

    Nocardicin A is a monocyclic β-lactam isolated from the actinomycete Nocardia uniformis that shows moderate antibiotic activity against a broad spectrum of Gram-negative bacteria. The monobactams are of renewed interest due to emerging Gram-negative strains resistant to clinically available penicillins and cephalosporins. Like isopenicillin N, Nocardicin A has a tripeptide core of non-ribosomal origin. Paradoxically, the Nocardicin A gene cluster encodes two non-ribosomal peptide synthetases (NRPSs), NocA and NocB, predicted to encode five modules pointing to a pentapeptide precursor in Nocardicin A biosynthesis, unless module skipping or other nonlinear reactions are occurring. Previous radiochemical incorporation experiments and bioinformatic analyses predict the incorporation of p-hydroxy-l-phenylglycine (l-pHPG) into positions 1, 3, and 5 and l-serine into position 4. No prediction could be made for position 2. Multidomain constructs of each module were heterologous expressed in Escherichia coli for d...

  • Non-ribosomal Propeptide Precursor in Nocardicin A Biosynthesis Predicted from Adenylation Domain Specificity Dependent on the MbtH Family Protein NocI
    2013
    Co-Authors: Jeanne M Davidsen, David M Bartley, Craig A Townsend
    Abstract:

    Nocardicin A is a monocyclic β-lactam isolated from the actinomycete Nocardia uniformis that shows moderate antibiotic activity against a broad spectrum of Gram-negative bacteria. The monobactams are of renewed interest due to emerging Gram-negative strains resistant to clinically available penicillins and cephalosporins. Like isopenicillin N, Nocardicin A has a tripeptide core of non-ribosomal origin. Paradoxically, the Nocardicin A gene cluster encodes two non-ribosomal peptide synthetases (NRPSs), NocA and NocB, predicted to encode five modules pointing to a pentapeptide precursor in Nocardicin A biosynthesis, unless module skipping or other nonlinear reactions are occurring. Previous radiochemical incorporation experiments and bioinformatic analyses predict the incorporation of p-hydroxy-l-phenylglycine (l-pHPG) into positions 1, 3, and 5 and l-serine into position 4. No prediction could be made for position 2. Multidomain constructs of each module were heterologous expressed in Escherichia coli for determination of the adenylation domain (A-domain) substrate specificity using the ATP/PPi exchange assay. Three of the five A-domains, from modules 1, 2, and 4, required the addition of stoichiometric amounts of MbtH family protein NocI to detect exchange activity. On the basis of these analyses, the predicted product of the NocA and NocB NRPSs is l-pHPG–l-Arg–d-pHPG–l-Ser–l-pHPG, a pentapeptide. Despite being flanked by non-proteinogenic amino acids, proteolysis of this pentapeptide by trypsin yields two fragments from cleavage at the C terminus of the l-Arg residue. Thus, a proteolytic step is likely involved in the biosynthesis of Nocardicin A, a rare but precedented editing event in the formation of non-ribosomal natural products that is supported by the identification of trypsin-encoding genes in N. uniformis

Stanislav Gobec - One of the best experts on this subject based on the ideXlab platform.

  • in silico design and enantioselective synthesis of functionalized monocyclic 3 amino 1 carboxymethyl β lactams as inhibitors of penicillin binding proteins of resistant bacteria
    Chemistry: A European Journal, 2018
    Co-Authors: Lena Decuyper, Sari Deketelaere, Lore Vanparys, Marko Jukic, Izidor Sosic, Eric Sauvage, Ana Maria Amoroso, Olivier Verlaine, Bernard Joris, Stanislav Gobec
    Abstract:

    As a complement to the renowned bicyclic beta-lactam antibiotics, monocyclic analogues provide a breath of fresh air in the battle against resistant bacteria. In that framework, the present study discloses the in silico design and unprecedented ten-step synthesis of eleven Nocardicin-like enantiomerically pure 2-{3-[2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido]-2-oxoazetidin-1-yl}acetic acids starting from serine as a readily accessible precursor. The capability of this novel class of monocyclic 3-amino-beta-lactams to inhibit penicillin-binding proteins (PBPs) of various (resistant) bacteria was assessed, revealing the potential of alpha-benzylidenecarboxylates as interesting leads in the pursuit of novel PBP inhibitors. No deactivation by representative enzymes belonging to the four beta-lactamase classes was observed, while weak inhibition of class C beta-lactamase P99 was demonstrated.

David M Bartley - One of the best experts on this subject based on the ideXlab platform.

  • non ribosomal propeptide precursor in Nocardicin a biosynthesis predicted from adenylation domain specificity dependent on the mbth family protein noci
    Journal of the American Chemical Society, 2013
    Co-Authors: Jeanne M Davidsen, David M Bartley, Craig A Townsend
    Abstract:

    Nocardicin A is a monocyclic β-lactam isolated from the actinomycete Nocardia uniformis that shows moderate antibiotic activity against a broad spectrum of Gram-negative bacteria. The monobactams are of renewed interest due to emerging Gram-negative strains resistant to clinically available penicillins and cephalosporins. Like isopenicillin N, Nocardicin A has a tripeptide core of non-ribosomal origin. Paradoxically, the Nocardicin A gene cluster encodes two non-ribosomal peptide synthetases (NRPSs), NocA and NocB, predicted to encode five modules pointing to a pentapeptide precursor in Nocardicin A biosynthesis, unless module skipping or other nonlinear reactions are occurring. Previous radiochemical incorporation experiments and bioinformatic analyses predict the incorporation of p-hydroxy-l-phenylglycine (l-pHPG) into positions 1, 3, and 5 and l-serine into position 4. No prediction could be made for position 2. Multidomain constructs of each module were heterologous expressed in Escherichia coli for d...

  • Non-ribosomal Propeptide Precursor in Nocardicin A Biosynthesis Predicted from Adenylation Domain Specificity Dependent on the MbtH Family Protein NocI
    2013
    Co-Authors: Jeanne M Davidsen, David M Bartley, Craig A Townsend
    Abstract:

    Nocardicin A is a monocyclic β-lactam isolated from the actinomycete Nocardia uniformis that shows moderate antibiotic activity against a broad spectrum of Gram-negative bacteria. The monobactams are of renewed interest due to emerging Gram-negative strains resistant to clinically available penicillins and cephalosporins. Like isopenicillin N, Nocardicin A has a tripeptide core of non-ribosomal origin. Paradoxically, the Nocardicin A gene cluster encodes two non-ribosomal peptide synthetases (NRPSs), NocA and NocB, predicted to encode five modules pointing to a pentapeptide precursor in Nocardicin A biosynthesis, unless module skipping or other nonlinear reactions are occurring. Previous radiochemical incorporation experiments and bioinformatic analyses predict the incorporation of p-hydroxy-l-phenylglycine (l-pHPG) into positions 1, 3, and 5 and l-serine into position 4. No prediction could be made for position 2. Multidomain constructs of each module were heterologous expressed in Escherichia coli for determination of the adenylation domain (A-domain) substrate specificity using the ATP/PPi exchange assay. Three of the five A-domains, from modules 1, 2, and 4, required the addition of stoichiometric amounts of MbtH family protein NocI to detect exchange activity. On the basis of these analyses, the predicted product of the NocA and NocB NRPSs is l-pHPG–l-Arg–d-pHPG–l-Ser–l-pHPG, a pentapeptide. Despite being flanked by non-proteinogenic amino acids, proteolysis of this pentapeptide by trypsin yields two fragments from cleavage at the C terminus of the l-Arg residue. Thus, a proteolytic step is likely involved in the biosynthesis of Nocardicin A, a rare but precedented editing event in the formation of non-ribosomal natural products that is supported by the identification of trypsin-encoding genes in N. uniformis